Project Summary/Abstract Physical Sciences Inc. (PSI) proposes to develop a handheld two-photon microscopy (TPM) probe that will enable in vivo diagnosis and screening of various diseases, including cancers. This novel, clinically usable TPM instrument will provide three-dimensional (3-D) sub-micron resolution images of tissue pathology in situ. The images generated based on the two-photon excitation signals of intrinsic bio-molecules, such as the two-photon excited fluorescence (TPEF) of endogenous fluorophores and the second-harmonic generation (SHG) of collagen, will render important morphological and molecular information specific to various pathologies. Noninvasive optical biopsy based on two-photon microscopy is considered as the next-generation technology for disease diagnosis and has already been investigated in many pre-clinical studies. However, a suitable clinical TPM instrument has not yet been demonstrated, which is largely due to the technical and cost barriers in developing such a sought-after system. These include difficulty in transmitting high-power broadband femtosecond pulses from the bulky laser to a hand-held probe through a flexible fiber cord; challenges in miniaturizing the imaging probe with no sacrifice of imaging performance; and the high cost of the ultrafast laser source needed for efficient excitation of the nonlinear optical processes. Therefore, this R&D program directly addresses these barriers to widespread clinical use of TPM for noninvasive pathology. A laboratory prototype will be developed in the Phase I to demonstrate feasibility, while further optimization, advanced engineering and clinical testing will be performed in Phase II. Innovative technologies, including the development of a specialty fiber module for delivery of high-power ultrashort pulses and generation of a coherent supercontinuum (SC) based on the fiber-delivered laser pulses, as well as novel technological approaches for building a compact light-weight imaging probe, are being proposed. This will be possible by leveraging the expertise of a multidisciplinary team of investigators from PSI, with a proven track record on developing the next generation clinical devices, a leading research group from University of Illinois at Urbana-Champaign, and experienced clinical researchers and pathologists from Memorial Sloan Kettering Cancer Center.